Compositions and method for treating precancerous virus infection and prevention of cancer

ABSTRACT

An anti-cancer composition having biocompatible materials, which can selectively exploit chemical variations between normal cells and cancer cells to inhibit or prevent the proliferation of cancerous cells and methods of use.

TECHNICAL FIELD

The present disclosure relates to pharmaceutical compositions andmethods of use for treating cancer in mammals.

BACKGROUND

All patents, scientific articles, and other documents mentioned hereinare incorporated by reference as if reproduced in full below. Cancer isthe rapid and uncontrolled proliferation of new cells within a body, andis a leading cause of death in animals, including humans. Thisproliferation far exceeds the normal level of apoptosis, thephysiological process essential to normal development and homeostasis ofmulticellular organisms. (Stellar, Science 267:1445-1449 (1995)).

Chemotherapy, often used in conjunction with radiation treatments andsurgery, is a standard cancer treatment used today. Chemotherapy isgenerally understood to mean medications or drugs that destroy cancercells. Presently, there are over one hundred drugs used in variouscombinations to treat cancer. (The American Cancer Society, ConsumersGuide to Cancer Drugs, Jones and Bartlett Publishers, (2000)). “Allthese drugs have one characteristic in common. They work because they'repoisons.” (Moss, Questioning Chemotherapy, Equinox Press, pg. 77,(2000)). Chemotherapeutic agents are highly toxic and typically havenarrow therapeutic indices. These agents exhibit little specificity formalignant cells, and they cannot discriminate effectively between normaland malignant cells. Consequently, all cells and tissues, and especiallyrapidly proliferating cells, such as the bone marrow cells, thespermatogonia, and the gastrointestinal crypt epithelium cells, are veryvulnerable. (Baquiran, Cancer Chemotherapy Handbook, Lippincott, pg. 85(2001)). Moreover, the side effects of chemotherapy can be horrific, asis well known to those of skill in the art and to those unfortunateenough to have the art practiced upon them. (The American CancerSociety, Consumers Guide to Cancer Drugs, Jones and Bartlett Publishers,(2000)). See also, (Baquiran, Cancer Chemotherapy Handbook, Lippincott,p 85 (2001)); (Chu & Devita, Physicians' Cancer Chemotherapy DrugManual, 2003, Jones and Bartlett Publishers, (2003)); (Lance Armstrong,It's Not About the Bike, Berkley Publishing, (2000)), (King, King andPearlroth, Cancer Combat, Bantam Books, (1998)); (Rich, The Red Devil,Three Rivers Press, (1999)); and (Marchione, Hopes in cancer drugdashed, Milwaukee Journal Sentinel, May 22, (2002)). Current cancertreatments including chemotherapy do not generally work well with solidtumors. (Moss, Questioning Chemotherapy, Updated Edition, Equinox Press,2000:18) and (Masters and Koberle, in Curing Metastatic Cancer: Lessonsfrom Testicular Germ-Cell Tumours, Nature Reviews, 3(7) (July 2003)).

Resistance can develop to chemotherapeutic agents, causing the agents towork for some types of cancer, but not for others, or not work at all.Resistance has been demonstrated to every single chemotherapeutic agentever developed. This resistance may be innate, acquired or emergentresistance. (Chu & Devita; Physicians' Cancer Chemotherapy Drug Manual.2003, Jones and Bartlett Pub. (2003)). In addition, it has been commonlyassumed that combining chemotherapeutic agents will result in regimenswith superior response rates. However, a study demonstrated thatchemotherapy agents, used either in sequence or in combination formetastatic breast cancer, provided equivalent results with regard tosurvival and quality of life was measured. (Sledge, et al., Phase III,Trial of Doxorubicin, paclitaxel, and the combination of doxorubicin andpaclitaxel as front-line chemotherapy for metastatic breast cancer: anintergroup trial, J. of Clin. Oncology, 21 (4):588-592 (February,2003)).

Additionally, a study utilizing four of the newer chemotherapy regimensand drugs produced a two-year survival rate of 11% and substantialtoxicity. The response and survival rate did not differ significantlyamongst the four groups treated with the different regimens for advancednon-small-cell lung cancer. (Schiller, et al., Comparison of FourChemotherapy Regimens for Advanced Non-Small-Cell Lung Cancer, The N.Eng. J. of Med., 346(2):92-98 (January, 2002)).

Cancer cells are well known to have a higher glucose uptake andmetabolism, and the resulting enhanced glycolysis can serve as anindication of a malignant transformation. (Mehvar, Dextrans for targetedand sustained delivery of therapeutic and imaging agents, J. ofControlled Release, 69:1-25 (2000)); (Essner, et al., Advances in FDGPET Probes in Surgical Oncology, Cancer Jour. 8:100-108 (2002)). Cancercells utilize and metabolize glucose at high rates, (even in thepresence of high oxygen concentrations) forming mostly lactate.(Warburg, O., On The Origin of Cancer Cells, Science 123 (3191): 309-314(February, 1956)). Lactate, therefore, is detected in cancer cells atmuch higher levels than in the corresponding normal tissues.(Rivenzon-Segal, et. al., Glycolysis as a metabolic marker in orthotopicbreast cancer, monitored by in vivo 13C MRS, Amer. J. Phys.Endocrinology Metabolism, 283: E623-E630 (2002); See also, (Lee andPedersen, Glucose Metabolism in Cancer, J. of Biol. Chem. 278(42):41047-41058 (October, 2003)); (Gatenby and Gawlinski, Theglycolysis phenotype in carcinogenesis and tumor invasion: insightsthrough mathematical models, Cancer Res., 63(14):3847-54 (July 2003));(Degani, The American Society of Clinical Oncology, Intn'l J. of Cancer,107:177-182 (November, 2003)); (Warburg, O. The Prime Cause andPrevention of Cancer, Konrad Triltsch, p 6. (1969)). Glucose typicallyenters most cells by facilitated diffusion through one of a family ofglucose transporters. (Katzung, Basic & Clinical Pharmacology, McGrawHill Co. Inc., pg. 715 (2001)). Glucose forms that are incompatible withthese transporters can be taken in by phagocytosis, also known asendocytosis, either by a cell of the phagocytic system or a cellassociated with a tissue. The phagocytic system, also known as thereticuloendothelial system (“RES”), or the mononuclear phagocyte system(“MPS”), is a diffuse system, which includes the fixed macrophages oftissues, liver, spleen, lymph nodes and bone marrow, along with thefibroblastic reticular cells of hemotopoietic tissues.

Glucose initiates, promotes, drives and amplifies the growth andmetastasis of tumor cells. Anaerobic glycolosis favored by tumor cells,is a very inefficient and primitive process to produce ATP, requiringprodigious amounts of glucose. Thus, the scientific community iscurrently working on ways to deprive tumor cells of glucose. (Van Danget al, The Proc. of the Nat'l Acad. of Sci. 95:1511-1516 (1998)).(Pedersen, Inhibiting glycolysis and oxidative phosphorylation. 3-BrPAabolishes cell ATP production, Reuters News, (Jul. 18, 2002)). An invivo murine study on xenograft models of human osteosarcoma andnon-small cell lung cancer found that the glycolytic inhibitor2-deoxy-D-glucose in combination with adriamycin or paxlitaxel, resultedin significant slower tumor growth. (Maschek, et al., 2-deoxy-D-glucoseincreases the efficacy of adriamycin and paclitaxel in humanosteosarcoma and non-small cell lung cancers in vivo, Cancer Res.,64(1):31-34 (2004)). In addition, positive clinical results have beenfound with the anti-cachexia drug, hydrazine sulfate, which inhibitsneoglucogenesis. (Moss, Cancer Therapy, Equinox Press, p 316 (1992)).Many dietary modifications directed at depriving cancer cells of glucosehave also been studied. (Quillin, Beating Cancer with Nutrition,Nutrition Times Press, p 225 (1998)); (Quillin, Cancer's Sweet Tooth,Nutrition Science News, (April 2000)); and (Hauser & Hauser,Cancer-Treating Cancer with Insulin Potentiation Therapy, Beulah LandPress, (2001)).

Copper (Cu), is an essential trace element, and necessary for life inorganisms ranging from bacteria to mammals. Copper promotes and is anessential co-factor for angiogenesis, a requirement for the growth ofcancer, especially solid tumors. (Brewer, Handbook of CopperPharmacology and Toxicology, Humana Press, Chap. 27, (2002)); (Brem,Angiogenesis and Cancer Control: From Concept to Therapeutic Trial,Cancer Control Jour., 6 (5):436-458 (1999). Since angiogenesis isgenerally not required in adults, the inhibition of angiogenesis throughcopper removal, copper reduction therapy, or copper withholding, hasbeen explored as a possible mechanism for inhibiting further tumorgrowth. (Brewer, supra); See, also U.S. Pat. No. 6,703,050 of Brewer etal. Tumors of many types have a great need for copper and sequestercopper, because copper is an essential cofactor for angiogenesis andproliferation. (Brewer. Copper Control as an Antiangiogenic AnticancerTherapy: Lessons from Treating Wilson's Disease, Exp. Bio. and Med.,226(7):665-673 (2001)). Because of this avidity for copper, and effectsof copper promoting tumor initiation, growth and metastasis, thescientific community continues to develop methods and pharmaceuticals ofwithholding copper from tumor cells. (Brem, supra); (Brewer, supra);(Brewer, et al., Treatment of Metastatic Cancer with Tetrathiomolybdate,an Anticopper, Antiangiogenesis Agent: Phase I Study, Clin. Cancer Res.,6:1-10 (2000)); (Redman, Phase II Trial of Tetrathiomolybdate inPatients with Advanced Kidney Cancer, Clin. Cancer Res., 9:1666-1672(2003)); (Pan, et al., Copper Deficiency Induced by TetrathiomolybdateSuppresses Tumor Growth and Angiogenesis, Cancer Res., 62:4854-4859(2002)); (Teknos, et al., Inhibition of the Growth of Squamous CellCarcinoma by Tetrathiomolybdate-Induced Copper Suppression in a MurineModel, Arch. of Otolaryngology: Head And Neck Surgery, Oncolink CancerNews, Reuters, 129:781-785 (2003)); (Yoshiji, et al., The CopperChelating Agent, trientine, suppresses tumor development andangiogenesis in the murine heptatocellular carcinoma cells, Int'l J. ofCancer, 94:768-773 (December, 2001); (Yoshiji, et al., The copperchelating agent. Trientine attentuates liver enzymes-alteredpreneoplastic lesions in rats by angiogenesis suppression, OncologyRep., 10(5):1369-73 (2003)); (Brem, et al., Penicillamine and Reductionof Copper for Angiosuppressive Therapy of Adults with Newly DiagnosedGlioblastoma, H. Lee Moffitt Cancer Center & Research Inst., (1999));(Sagripanti and Kraemer, Site-specific Oxidative DNA Damage atPolyguanosines Produced by Copper Plus Hydrogen Peroxide, J. of Biol.Chem., 264(3):1729-1734 (1989)).

Copper may also promote cancer growth in ways such as damaging DNA.(Sagripanti, supra (1999)). The destructive activity of copper in a cellincludes binding to DNA, cleaving DNA, in the presence of reducants andhydrogen peroxides, nonspecific disruption of cellular function, and thegeneration of free hydroxyl radicals through Haber-Weiss reactions.(Theophanides, et al., Copper and Carcinogenesis, Critical Reviews InOncology/Hematology, 42:57-64 (2002)). Copper also plays a role in theformation of reactive oxygen species (“ROS”). (Sagripanti, DNA DamageMediated by Metal Ions with Special Reference to Copper and Iron, Met.Ions Biol. Syst. 36:179-209(1999)).

The use of copper has also been disclosed for the treatment of cancer ina number of U.S. patents as well: U.S. Pat. No. 4,952,607 disclosescopper complexes exhibiting super oxide dismutase-like activity inmammalian cells; U.S. Pat. No. 5,124,351 discloses the use of copperchelate of nitrilotriacetic acid or a copper chelate ofbis-thiosemicarbazone; U.S. Pat. No. 5,632,982 discloses the use ofcopper chelates in conjunction with a surface membrane protein receptorinternalizing agent, particularly TNF for use against target cells; andU.S. Pat. No. 6,706,759 discloses the use of dithiocarbamate derivativesand copper.

It is also known that a quantitative difference exists between cancercells and normal cells with respect to iron requirements, becauseenhanced acquisition of iron initiates, promotes, and amplifies thegrowth, and metastasis, of tumor cells. Iron is an essential transitionmetal for a large number of biological processes ranging from oxygentransport through DNA synthesis and electron transport. Iron is alsoinvolved in carcinogenic mechanisms, which include the generation of DNAdamaging reactive oxygen species, and the suppression of host celldefenses. (Desoize, B., Editor, Cancer in Metals and Metal Compounds:Part I—Carcinogenesis, Critical Reviews In Oncology/Hematology, 42:1-3(2002)); (Galaris, et al., The Role of Oxidative Stress in Mechanisms ofMetal-induced Carcinogenesis, Critical Reviews In Oncology/Hematology,42:93-103 (2002)); (Weinberg, Cancer and Iron: a Dangerous Mix, IronDisorders Insight, 2(2):11 (1999)); (Weinberg, The Development ofAwareness of the Carcinogenic Hazard of Inhaled Iron, Oncology Res.11:109-113 (1999)); (Weinberg, Iron Therapy and Cancer, Kidney Int'l,55(60): S131-134 (1999)); (Weinberg, The Role of Iron in Cancer, Euro.J. Cancer Prevention, 5:19-36, (1996)); (Weinberg, Iron in NeoplasticDisease, Nutrition Cancer, 4(3):223-33 (1993)); (Stevens, et al., BodyIron Stores and the Risk of Cancer, N. Eng. J. of Med.,319(16):1047-1052 (1988)).

A number of pharmaceuticals have been developed to control and restrictthe supply of iron to tumor cells using different approaches, includingintracellular iron-chelating agents for withdrawal of the metal, use ofgallium salts to interfere with iron uptake, and utilization ofmonoclonal antibodies to transferrin receptors on tumors to block theuptake of iron. For example, U.S. Pat. No. 6,589,96, incorporated hereinin its entirety, teaches the use of iron chelators as chemotherapeuticagents against cancer to deprive cancer cells of iron. See also, (Kwok,et al., The Iron Metabolism of Neoplastic Cells: alterations thatfacilitate proliferation?, Crit. Rev. In Oncology/Hematology, 42:65-78(2002), discloses tumor cells express high levels of the transferrinreceptor 1 (TFR1) and internalize iron (Fe) from transferrin (TF) at atremendous rate); (Desoize, B. Editor, Cancer and Metals and MetalCompounds, Part II—Cancer Treatment, Crit. Rev. In Oncology/Hematology,42:213-215 (2002)); (Collery, et al., Gallium in Cancer Treatment, Crit.Rev. In Oncology/Hematology, 42:283-296 (2002)); (Weinberg, Developmentof Clinical Methods of Iron Deprivation for Suppression of Neoplasticand Infectious Diseases, Cancer Investigation, 17(7):507-513 (1999));(Weinberg, Human Lactoferrin: a Novel Therapeutic with Board SpectrumPotential, Pharmacy & Pharmacology, 53 (October 2001)); (Richardson,Iron Chelators as therapeutic agents for the Treatment of Cancer, Crit.Rev. In Oncology/Hematology, 42:267-281 (2002)).

When an iron dextran complex is administered to the blood system, thecellular toxicity of iron is blocked by the dextran sheath or shell indoses above or below the rate of clearance of the RES system. (Lawrence,Development and Comparison of Iron Dextran Products, J. of Pharm. Sci. &Tech., 52(5):190-197(1998)); (Cox, Structure of an iron-dextran complex,J. of Pharma & Pharmac, 24:513-517 (1972)); (Henderson & Hillman,Characteristics of Iron Dextran Utilization in Man, Blood, 34(3):357-375(1969)); U.S. Pat. No. 5,624,668). Iron dextran can remain in the plasmaand traffic throughout the body for weeks inertly, while being removedfrom the plasma by the phagocytic system and cancer cells.

Copper and iron are essential micronutrients for all organisms becauseof their function as co-factors in enzymes that catalyze redox reactionsin fundamental metabolic processes. (Massaro, editor, Handbook of CopperPharmacology and Toxicity, Humana Press, 2002, Chapter 30, p 481).Studies have shown synergistic interactions between iron and copper,which result in a significant increase in utilization of iron ascompared to the utilization found with iron only compounds. (Massaro,Chap. 30, supra). To bind iron to the plasma protein transferrin,oxidation is required from Fe2+ to Fe3+. The oxidation may be mediatedby multicopper ferroxidases, hephaestin or ceruloplasmin. Hephaestin mayact together with Ferroportin1 at the surface of enterocytes to oxidizeFe2+ to Fe3+ prior to export into blood plasma for loading ontotransferrin. An additional important role of ceruloplasmin is themobilization of iron from tissues such as the liver where ceruloplasminis synthesized. The ceruloplasmin can contain six copper atoms, issecreted from the liver, and can carry at least 95% of total serumcopper for delivery to tissues. In addition, ceruloplasmin, via itsferroxidase activity, mediates iron release from the liver, also fordelivery to tissues. Thus, both copper and iron support thehematopoietic system, especially red blood cell formation. Each isessential for the formation of red blood cells.

The American Cancer Society report, Cancer Facts and Figures 2003,discloses that “cancer is a group of diseases characterized byuncontrolled growth and spread of abnormal cells . . . . About 1,334,100new cancer cases are expected to be diagnosed in the United States in2003, with 556,500 cancer deaths expected in 2003.” The presentinvention includes, but is not limited to, the treatment of thesecancers disclosed in Cancer Facts and Figures 2003, page 4, supra, suchas, Oral Cavity and Pharynx, Digestive System, Respiratory System, Bonesand Joints, Soft Tissue, Skin, Breast, Genital System, Urinary System,Eye and Orbit, Brain and Other Nervous System, Endocrine System,Lymphoma, Multiple Myeloma, Leukemia, and Other Unspecified PrimarySites. Treatment with the present invention also includes basal andsquamous cell skin cancers and in situ carcinomas, Hyper ProliferativeDisorders, myelodysplasia disorders and Plasma Cell Dyscrasias, which ischaracterized by an increase in plasma cells in the bone marrow, oruncommonly, other tissue. A description of these clinical abnormalitiesis disclosed by Markman, M. D. in Basic Cancer Medicine, W. B. SaundersCo., p. 103, (1997).

It would be advantageous to develop an effective chemotherapeutic agentwhich employs biocompatible materials, materials which feed every cellin the body, to effectuate cell death, at minimum, prevent cancer cellreplication, and avoid classic and numerous deadly chemotherapeutic sideeffects. Such a therapeutic agent would avoid the issues of tissueresistance and lack of specificity that are caused by manypharmaceuticals, thereby destroying or disabling many previouslyunmanageable cancers without debilitating or killing the patient.

SUMMARY OF THE INVENTION

This disclosure relates to a Composition having anti-cancer propertiesand methods of use in mammals. A chemical Composition for use as apharmaceutical for treating cancer of a biologically acceptable coppercompound and may include other components such as iron, which istransported to afflicted cells in a pharmaceutical acceptable carrier.For example, the compound may be formed of a core of at leastbiologically acceptable copper compound which may be encapsulated with asheath that surrounds or coats the copper compound core and preventsimmediate chemical interaction of the core with the surroundingenvironment. The Composition is combined with a pharmaceuticallyacceptable carrier for administration to patients and may be used aloneor in conjunction with conventional cancer treatments.

Also disclosed is a method for treating cancers by administering theComposition having a biologically acceptable copper compound core, witha sheath encapsulating the copper compound core, and a pharmaceuticalcarrier to the patient. The patient is monitored regularly to determinethe level and/or presence of the cancer. The Composition may bere-administered at intervals determined to be medically necessary by thephysician, based on the results of the monitoring.

Without limitation, these and other objects, features, and advantages ofthe present invention, will become apparent to those with skill in theart after review of the following detailed description of the disclosedembodiments and the appended drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the release of ROS by HT29 human-colonadenocarcinoma cells by iron dextran alone and the Composition alone andin combination after 24 hours preincubation.

FIG. 2A is a graph of the concentration of the Composition alone plottedagainst NCI-H23 lung cells mean percent inhibition.

FIG. 2B is a chart of NCI-H23 lung cells dose response with control, theComposition alone, Base Compound alone and doxorubicin alone.

FIG. 2C is a chart of NCI-H23 lung cells with the Composition alone,with the IC₅₀ value.

FIG. 2D is a graph of the concentration of the Composition plus BaseCompound plotted against NCI-H23 lung cells mean % inhibition.

FIG. 2E is a chart of NCI-H23 lung cells with control, the Compositionplus Base Compound, Base Compound alone and doxorubicin alone.

FIG. 2F is a chart of NCI-H23 lung cells with the Composition plus BaseCompound, with the IC₅₀ value.

FIG. 3A is a graph of the concentration of the Composition alone plottedagainst NCI-H460 lung cells mean % inhibition.

FIG. 3B is a chart of NCI-H460 lung cells with control, the Compositionalone, Base Compound alone, and doxorubicin alone.

FIG. 3C is a chart of NCI-H460 lung cells with the Composition along,with the IC₅₀ value.

FIG. 3D is a graph of the concentration of the Composition plus BaseCompound plotted against NCI-H460 lung cells mean percent inhibition.

FIG. 3E is a chart of NCI-H460 lung cells dose response with control,the Composition plus Base Compound, Base Compound alone and doxorubicinalone.

FIG. 3F is a chart of NCI-H460 lung cells with the Composition plus BaseCompound, with the IC₅₀ value.

FIG. 4A is a graph of the concentration of the Composition alone plottedagainst MCF7 mammary cells mean percent inhibition.

FIG. 4B is a chart of MCF7 mammary cells dose response with control, theComposition alone, Base Compound alone and doxorubicin alone.

FIG. 4C is a chart of MCF7 mammary cells with the Composition alone,with the IC₅₀ value.

FIG. 4D is a graph of the concentration of the Composition plus BaseCompound plotted against MCF7 mammary cells mean % inhibition.

FIG. 4E is a chart of MCF7 mammary cells with control, the Compositionplus Base Compound, Base Compound alone and doxorubicin alone.

FIG. 4F is a chart of MCF7 mammary cells with the Composition plus BaseCompound, with the IC₅₀ value.

FIG. 5A is a graph of the concentration of the Composition alone plottedagainst ZR-75-1 mammary cells mean percent inhibition.

FIG. 5B is a chart of ZR-75-1 mammary cells dose response with control,the Composition alone, Base Compound alone and doxorubicin alone.

FIG. 5C is a chart of ZR-75-1 mammary cells with the Composition alone,with the IC₅₀ value.

FIG. 5D is a graph of the concentration of the Composition plus BaseCompound plotted against ZR-75-1 mammary cells mean % inhibition.

FIG. 5E is a chart of ZR-75-1 mammary cells alone with control, theComposition plus Base Compound, Base Compound alone and doxorubicinalone.

FIG. 5F is a chart of ZR-75-1 mammary cells with the Composition plusBase Compound, with the IC₅₀ value.

FIG. 6A is a graph of the concentration of the Composition alone plottedagainst PC-3 prostate cells mean percent inhibition.

FIG. 6B is a chart of PC-3 prostate cells dose response with control,the Composition alone, Base Compound alone and doxorubicin alone.

FIG. 6C is a chart of PC-3 prostate cells with the Composition alone,with the IC₅₀ value.

FIG. 6D is a graph of the concentration of the Composition plus BaseCompound plotted against PC-3 prostate cells mean % inhibition.

FIG. 6E is a chart of PC-3 prostate cells with control, the Compositionplus Base Compound, Base Compound alone and doxorubicin alone.

FIG. 6F is a chart of PC-3 prostate cells with the Composition plus BaseCompound, with the IC₅₀ value.

FIG. 7A is a graph of the concentration of the Composition alone plottedagainst DLD-1 colon cells mean percent inhibition.

FIG. 7B is a chart of DLD-1 colon cells dose response with control, theComposition alone, Base Compound alone and doxorubicin alone.

FIG. 7C is a chart of DLD-1 colon cells with the Composition alone, withthe IC₅₀ value.

FIG. 7D is a graph of the concentration of the Composition plus BaseCompound plotted against DLD-1 colon cells mean % inhibition.

FIG. 7E is a chart of DLD-1 colon cells with control, the Compositionplus Base Compound, Base Compound alone and doxorubicin alone.

FIG. 7F is a chart of DLD-1 colon cells with the Composition plus BaseCompound, with the IC₅₀ value.

FIG. 8A is a graph of the concentration of the Composition alone plottedagainst OVCAR-3 ovarian cells mean percent inhibition.

FIG. 8B is a chart of OVCAR-3 ovarian cells dose response with control,the Composition alone, Base Compound alone and doxorubicin alone.

FIG. 8C is a chart of OVCAR-3 ovarian cells with the Composition alone,with the IC₅₀ value.

FIG. 8D is a graph of the concentration of the Composition plus BaseCompound plotted against OVCAR-3 ovarian cells mean % inhibition.

FIG. 8E is a chart of OVCAR-3 ovarian cells with control, theComposition plus Base Compound, Base Compound alone and doxorubicinalone.

FIG. 8F is a chart of OVCAR-3 ovarian cells with the Composition plusBase Compound, with the IC₅₀ value.

FIG. 9A is a graph of the concentration of the Composition alone plottedagainst CAKI-1 renal cells mean percent inhibition.

FIG. 9B is a chart of CAKI-1 renal cells dose response with control, theComposition alone, Base Compound alone and doxorubicin alone.

FIG. 9C is a chart of CAKI-1 renal cells with the Composition alone,with the IC₅₀ value.

FIG. 9D is a graph of the concentration of the Composition plus BaseCompound plotted against CAKI-1 renal cells mean % inhibition.

FIG. 9E is a chart of CAKI-1 renal cells with control, the Compositionplus Base Compound, Base Compound alone and doxorubicin alone.

FIG. 9F is a chart of CAKI-1 renal cells with the Composition plus BaseCompound, with the IC₅₀ value.

FIG. 10A is a graph of the concentration of the Composition aloneplotted against LOX IMVI melanoma cells mean percent inhibition.

FIG. 10B is a chart of LOX IMVI melanoma cells dose response withcontrol, the Composition alone, Base Compound alone and doxorubicinalone.

FIG. 10C is a chart of LOX IMVI melanoma cells with the Compositionalone, with the IC₅₀ value.

FIG. 10D is a graph of the concentration of the Composition plus BaseCompound plotted against LOX IMVI melanoma cells mean % inhibition.

FIG. 10E is a chart of LOX IMVI melanoma cells with control, theComposition plus Base Compound, Base Compound alone and doxorubicinalone.

FIG. 10F is a chart of LOX IMVI melanoma cells with the Composition plusBase Compound, with the IC₅₀ value.

FIG. 11A is a graph of the concentration of the Composition aloneplotted against SBN-75 CNS cells mean percent inhibition.

FIG. 11B is a chart of SBN-75 CSN cells dose response with control, theComposition alone, Base Compound alone and doxorubicin alone.

FIG. 11C is a chart of SBN-75 CNS cells with the Composition alone, withthe IC₅₀ value.

FIG. 11D is a graph of the concentration of the Composition plus BaseCompound plotted against SBN-75 CNS cells mean % inhibition.

FIG. 11E is a chart of SBN-75 CNS cells with control, the Compositionplus Base Compound, Base Compound alone and doxorubicin alone.

FIG. 11F is a chart of SBN-75 CNS cells with the Composition plus BaseCompound, with the IC₅₀ value.

FIG. 12A is a graph of the concentration of the Composition aloneplotted against CEM-SS Leukemic cells mean percent inhibition.

FIG. 12 B is a chart of the assayed toxicity values of the CEM-SSLeukemic cells data.

FIG. 12C provides the IC₅₀ of the CEM-SS Leukemic cells data.

FIG. 13A is a graph of the concentration of the Composition aloneplotted against CEM-SS leukemic cells mean percent inhibition.

FIG. 13 B is a chart of the assayed toxicity values of the CEM-SSLeukemic cell data.

FIG. 13C provides the IC₅₀ of the CEM-SS Leukemic cell data.

FIG. 14 is a table of the cell lines used and the results of thisdisclosure.

FIGS. 15 A, B, and C are portions of a table on the concentration of theequivalent of elemental iron, which was derived from iron dextran, foundin the monkey plasma over time.

FIG. 16 is a table of the single dose administrations of elemental iron,which was derived from iron dextran, found in the monkey plasma overtime.

DETAILED DESCRIPTION

This disclosure relates to a Composition which can selectively exploitchemical variations and requirements between normal cells and cancercells to inhibit and/or prevent the proliferation of cancerous cells inmammals. Most cancer treatments are unfocused and detrimentally affecthealthy cells as well as cancerous cells in contact with the treatmentbecause of a lack of specificity in traditional treatments. The abilityof the disclosed Composition to exploit these chemical differences andrequirements, and target cancer cells focuses the therapeutic agent tothe desired cells and limits effects on healthy cells of a mammal. Thedisclosed chemical Composition, therefore, provides a chemotherapeuticthat is less toxic with reduced side effects. This disclosure relates tothe addition of glucose, copper and iron compounds to cancer cells, cellproliferating diseases (such as pre-cancerous cells, psoriasis, and soon), hyper proliferative disorders, myelodysplasia disorders, plasmacell dyscrasias, solid tumors, liquid tumors, and metastatic diseases toshrink tumors by killing tumor cells and/or arresting their growth. TheComposition employs agents, which have been shown to be effectiveanti-cancer agents in the Examples below, although recurrently thesubject of research with respect to the withholding, restricting,limiting and modulating intended to block initiation, promotion, andgrowth of tumors and metastasis of cancer cells.

The Composition is comprised of, at least, nanoparticles of a fixedcopper compound core, or a fixed copper-iron compound core, or acombination of the two. These cores may be encapsulated, coated,adsorbed, complexed, or the like, with a protective sheath or jacketwhich also functions to target cancer cells. This sheath or jacket maybe any combination of materials, such as a glucose or liposome, and,optionally, the resulting glucose encapsulated core may be coated withliposomes. In another embodiment, the core may be encapsulated withdextran alone or any glucose or combination of sugar-based substances.Alternatively, a liposome encapsulated core may then be coated with anouter dextran sheath.

As transition metals, copper and iron can generate reactive oxygenspecies including hydroxyl radicals. It is widely recognized thattransition metals, including Cu⁺, Fe²⁺, Sn³⁺, Co²⁺ and Ni²⁺, have beendemonstrated to cause catalysis of free-radical reactions in biologicalsystems. Therefore, cancer cells can be destroyed by digestion andfragmentation, which can be achieved by oxidation by copper or iron,and/or catalyzed free-radical chemical reactions. The Cu²⁺ associateswith the guanine-cytosine base pairs of DNA to cause local free-radicaldamage to the DNA that is characteristic of attack by hydroxyl ion.Copper is a promoter of free-radical damage to lipids, proteins, andespecially to DNA and its base pairs. (Aruoma, Copper ion-dependentdamage to the base pairs in DNA in the presence of hydrogen peroxide,Biochem. Jour., 273: 601-4(1991)). In addition to the generation ofoxygen species, the transitional metals, copper and iron, may belimiting nutrients to the growth and replication of cancer cells inmammals, as has been demonstrated in many in vitro, mammalian studies.

Suitable copper compounds for use as the core are any biologicallyacceptable copper compounds which include, but are not limited to, anyfixed coppers including, cupric hydroxide, copper oxide, copperoxychloride, cupric carbonate basic, copper sulfate, copper sulfatebasic, cuprous oxide, cupric hydroxide-iron hydroxide, copper-ironoxide, cupric citrate, cupric glycinate, cupric gluconate, cupricphosphate, cuprobam, cupric salicylite, indigo copper, cupro-cupricsulfate, cuprous sulfate, cuprous sulfate hemihydrite, any of thenatural copper containing minerals such as cupric sulfate basic, theminerals brochantite, langite, malachite, azurite, cheesylite,cornetite, dihydyrite, libethenite, phosphorochalcite,pseudolibethenite, pseudo-malachite, tagilite, antlerite, covellite,marshite, cuprite, chalcocite, Rogojski's salt, brochantite,hydrocyanite, chalcanthtite, and the like, or any copper mineralsoccurring in nature such as nantokite or dolerophane and so on. Seealso, for examples of copper compounds, Merck's Manual 13^(th) ed.,Merck & Co. 2001, and Hawley's Condensed Chemical Dictionary 14^(th)ed., John Wiley & Sons, Inc. 2001. Copper hydroxide, a fixed copper, isa preferred compound to form the core. In another embodiment, the coremay also be composed of cupric hydroxide-iron hydroxide to provide asynergistic effect, which enhances the cellular toxicity of both thecopper and iron. In one embodiment, any biocompatible form of coppercompound that can cause catalysis of free-radical reactions inbiological systems may be used as a core metal for the disclosedComposition. A biologically acceptable copper compound as defined hereinis a copper compound, which may be used with and within a biologicalsystem with little or no detrimental effect, i.e. it does notappreciably alter or appreciably affect in any adverse way, thebiological system into which it is introduced.

In a further embodiment, a combination of copper oxide, copperhydroxide-iron hydroxide or another of the fixed coppers and iron, maybe used as a core to provide synergistic effects of the combination. Anybiocompatible iron compound may be used in conjunction with the coppercore, including without limitation, for example, Fe³⁺, and its salts,iron hydroxide, iron oxyhydroxide, iron oxide, iron glucose, ferriccitrate, Ferritin, ferrous fumarate, ferrous sulfate, and the like, toiron load the biological environment, including iron-saturated humanholotransferrin.

The nanoparticles of the disclosed Composition preferably can beencapsulated, surrounded, complexed, or adsorbed by, and bound to, atleast one sheath or coat that is preferably composed of a sugarsubstance, such as a glucose, a saccharide, a polysaccharide e.g.starch, cellulose, dextrans, alginides, chitosan, pectin, hyaluronicacid, pullulan (a bacterial polysaccharide), dextran, carboxyalkyldextran, carboxyalkyl cellulose and the like. These dextrans caninclude, for example, those disclosed by Mehvar, supra (2000); andRecent Trends in the Use of Polysaccharides for Improve Delivery ofTherapeutic Agents: Pharmacokinetic and Pharmacodynamic Perspectives,Curr. Pharm. Biotech. 4:283-302 (2003), and liposomes coated withdextran as disclosed by Moghimi, et al., Long-Circulating andTarget-Specific Nanoparticles: Theory to Practice, Pharm. Rev.,53(2):283-318 (2001)) both of which are incorporated herein in theirentirety. The sheath encoats, or encapsulates, the disclosedComposition's core and prevents chemical interaction of the core withthe surrounding environment, blocking the degradation of the core andthe emanation of the copper and/or iron from the copper compound, and/orthe copper-iron compound from the core. The thickness of the sheath maybe varied, if desired, by those skilled in the art. Because the sheathis composed primarily of a substance that is not necessarily recognizedby the body as foreign matter, the body is less likely to develop aresistance to the Composition. In one embodiment, the sheath can becomposed of dextran, also known as macrose, a high molecular weightpolysaccharide. Dextran is an ideal candidate for use as a sheathbecause it is often administered to mammals as a blood plasma substituteor expander, is generally not rejected by the mammalian system, and canremain in the plasma for an extended period of time. Other biocompatiblematerials for the formation of a polymeric shell, sheath, or jacket caninclude proteins, polypeptides, oligopeptides, polynucleotides,polysacchrides, lipids and so on. Additional sheath materials include,for example, those of U.S. Pat. No. 6,096,331; and U.S. Pat. No.6,506,405, incorporated herein in their entirety. Alternatively,combinations of two or more of the above named materials may be used toform the sheath.

In another embodiment, the disclosed Composition can be sheathed orencapsulated with a liposome coat. This liposome coat may be the solesheath encapsulating the core, or may be a second coat over one, or acombination, of the above named materials. PEG liposome polymer coatingshave been shown to reduce phagocytic system uptake and provide longresidence time according to research by the Alza Corporation, DeliveryTimes, Issues and Opportunities, Vol 2 (1), incorporated herein in itsentirety. Residence time in the plasma can be extended to periods of atleast several days to weeks after IV injection without releasing theencapsulated drug, which would lower the administration frequency of thedrug. See, e.g., U.S. Pat. No. 6,465,008; U.S. Pat. Pub.US2002/017271181; U.S. Pat. Pub. US2001/005118381; each of which isincorporated herein in its entirety.

Alternatively, the core may be transported to cell-specific sites withthe use of targeting agents or markers which may target cancer cells,cell proliferating diseases (such as pre-cancerous cells, psoriasis, andso on), solid tumors, liquid tumors, and metastatic diseases. Anytargeting agent or marker which can medicinally utilized within abiological system may be employed to actively transport the core to thespecific site of the cancer cells (See, for example, R. C. Juliano,Targeted Drug Delivery, Handbook of Experimental Pharmacology, Vol. 100,Ed. Born, G. V. R. et al., Springer Verlag). For example, a bindingmolecule to a cancerous cell surface site or cell surface receptor,surfactant, a ligand, an antibody, proteins, peptides, enzymes, specificchemical compounds, and so on, may be used as targeting agents ormarkers to target cancer cells. These targeting agents or markers may beused instead of, or in conjunction with, at least one sheathencapsulating the core.

The nanoparticle size of the entire disclosed Composition may beapproximately 1 nm to approximately 10,000 nm. In a more preferredembodiment, the particle size may be approximately 15 nm toapproximately 500 nm. A most preferred embodiment for particle size isapproximately 20 nm to approximately 200 nm.

Empty liposomes, which are devoid of drugs, may be co-administered oradministered before, during, or after the Composition itself to thepatient, to function as a decoy, placebo carrier, or redistributionagent with respect to the phagocytic system and allow the Composition toremain in the plasma for an extended period of time. The empty liposomedecoys, or placebo carriers, occupy the phagocytic system and alsoredistribute the disclosed composition away from clearance by cells inthe liver and in the spleen and thus concentrate the disclosedcomposition in the plasma for an extended period of time. Biocompatiblematerials used for polymeric shells may also be employed as decoys,alone or in combination with liposomes.

Iron dextran is also an exemplary example of a biocompatible ironcompound which iron loads tissues through at least two differentpathways, and works advantageously with the disclosed Composition as aredistribution agent. The first is phagocytosis by cancer cells throughan extended human plasma residence time. The second is increasing thetransferrin saturation through processing of the iron dextran throughthe phagocytic system. The intra-cellular metabolism of iron dextranwithin a tumor cell increases the acidity of the environment, whichfurther promotes the breakdown of the disclosed Composition. For thepurposes of this patent application, phagocytosis and endocytosis aredefined as the uptake of material, including particulate materials, intoa cell by the formation of a membrane vesicle, and are used herein asequivalent terms.

In one embodiment, the disclosed composition plus iron dextran plusempty liposomes may be added to the total parenteral nutrition (“TPN”)for the cancer patient. The disclosed composition includes essentialtrace elements of copper, and may include iron, as well as glucose,and/or liposomes, which are fats, to contribute to the patient's bodilyrequirements. Thus the Composition also provides an importantcontribution to the total parenteral nutrition of the patient.

In yet another embodiment, the Composition may be used with insulinpotentiation therapy (“IPT”), with or without iron dextran, to promotethe ingestion of these agents of the invention into the tumor cell.(Hauser & Hauser, Cancer-Treating Cancer with Insulin PotentiationTherapy. Beulah Land Press, p 267 (2001)).

Without being limited, held, or bound to any particular theory ormechanism of action, it is believed that the Composition, theredistribution agents, i.e., iron dextran with or without emptyliposomes, enters the system, traffics throughout the body as an inertentity, and is removed from the plasma by the phagocytic system and/orcancer cells. The Composition functions as a prodrug, it is inert in theplasma and active intracellularly within cancer cells. The Compositioncan remain in the mammal's plasma for a period of many days, dependingon the dosage levels, when used with a redistribution agent or placebocarrier. (It is known that iron-dextran can remain in the plasma forweeks, especially when doses are administered above the clearance rateof the phagocyte system. The processing of the iron dextran by thephagocytic system is rate limited to a daily maximum amount, leaving thebalance for future use.) The sheath may not be immediately recognized asforeign matter by the phagocytic system because it is a sugar-basedsubstance and is not rejected by the mammalian system, allowing theComposition to remain in circulation of the mammal for a longer periodthan most therapeutics, making it more likely to come into contact withtarget cells and providing more efficacy with fewer doses thantraditional chemotherapeutic agents. The Composition circulates, via anybiological pathway, throughout the body and may contact any cell type.For the most part, the phagocytic system takes up the Composition, as docancer cells which have a high affinity to phagocytize moleculesnecessary for proliferation, such as sugars. Normal, healthy cellsgenerally have very little interaction with the Composition. TheComposition that is taken up by the phagocytic system is processed, to alarge degree, through the liver in hepatocytes that store glucose, iron,and copper and are later released through their appropriate proteincarriers to feed and nurture cells of the body. Since sugars, copper,and iron are bodily requirements, well known to the phagocytic system,the phagocytic system is able to process, transport, store, or eliminatethem with little toxicity, while the Composition kills cancer cells andsimultaneously feeds and nourishes cells in the body.

When the Composition is phagocytized by cancer cells, or enters thecells by other means, the Composition is exposed to the cells' acidicenvironment, including lactic acid, caused by the anaerobic glycolysisprocess which is common to cancer cells. Any iron dextran that may bepresent in the cell also contributes to the acidity of the environmentduring the breakdown of the iron dextran compound. The sugar sheath ismetabolized and the core of the disclosed Composition breaks down underacidic conditions, generating at least free ions, free radicals, andreactive oxygen species (“ROS”). The free radicals taken together withthe free transition metal ions have cytotoxic effects on the cells andgenerate DNA-damaging free radicals and ROS. The free radicals and ROSprevent replication of the cell and, eventually, cause cell death. Incontrast, normal healthy cells generally process glucose aerobically,without lactic acid production. Therefore, if phagocytized by normalcells, the sheath is not readily broken down and the metal core remainssafely encapsulated in the sheath, which buffers the cellular toxicityof the core.

Copper is well known to those skilled in the art as a potent viricide.In vitro testing has shown that copper with hydrogen peroxide killssurrogate models of virtually every microorganism afflicting mammals.(See, Sagripanti, et al., Virus Inactivation by Copper or Iron Ionsalone and in the Presence of Peroxide, Applied and Environ. Microbio,59:12, 4374-4376 (1993); Sagripanti, Metal-based Formulations with HighMicrobicidal Activity, Applied and Environ. Microbio, 58:9, 3157-3162(1992)). The disclosed composition has also been shown effective as apotent viricide, and without being bound to a particular theory ormechanism, it is believed that the viricidal action functions asdescribed above to disrupt the viral DNA and rupture the viral envelope.The disclosed Composition can be useful to destroy those viruses knownto cause cancer, such as, for example, HBV and HCV for hepatocellularcarcinoma, HPV for cervical cancer, EBV (Epstein-Barr virus) forBurkitt's lymphoma, and HTLV 1 for a form of leukemia. Thus thedisclosed composition, with or without the addition of the iron-dextranbase, is active in the pre-cancerous stages, before the cells becomefully transformed. The disclosed composition may advantageously trafficthroughout the body, including the central nervous system and brain.

The administration of iron compositions and/or iron dextran compositionsmay be combined with the disclosed Composition to provide synergisticreactions between the copper and iron for enhanced cellular toxicity.The synergy between copper and iron is known in the art, and has beendescribed in the literature, see, for example, U.S. Pat. No. 5,202,353,incorporated herein in its entirety, which discloses use of thesynergistic affects of copper compositions and iron compositions for useas fungicides and bactericides. The iron compositions and/or irondextran compositions may also be administered to redistribute thedisclosed Composition and allow the Composition a longer residence timein the patient's plasma. Far higher dosages of iron dextran may beemployed, than that of elemental iron salts, for a greater cytotoxicity,and a protracted residence plasma time. The greater the iron level, thegreater the synergistic cytotoxicity of the Composition. Because it iswell known in the art that the phagocytic system removes the smallerparticles from the plasma circulation first, the combination of the irondextran with a smaller diameter than the Composition allows a protractedplasma residence time. The diameters of the iron dextran and the core ofthe disclosed Composition may be varied to manipulate the plasma time ofthese particles as desired. In one embodiment, the iron dextran can beadministered above the clearance level of the phagocyte system, whichcan serve as a decoy, placebo carrier, or redistribution agent to allowthe Composition to remain in the plasma for an extended period of time.(See, Henderson & Hillman, Characteristics of Iron Dextran Utilizationin Man, Blood, 34(3):357-375(1969)). This use of iron dextran at a doseabove the rate of clearance of the phagocyte system, to allow thedisclosed Composition to remain in the plasma for an extended period oftime, is known in the art as a redistribution (away from the liver andspleen to the plasma). Generally, smaller doses of iron dextran (50-500mg) are cleared within approximately 3 days, larger doses of irondextran (>500 mg), however, are cleared at a constant rate of 10-20mg/hr and are typically associated with increased plasma concentrationof iron dextran for as long as 3 weeks. Other agents which may serve asdecoys for the phagocytic system to redistribute the disclosedComposition to the plasma include, without limitation, pullulan, dextransulfate, empty liposomes, and those taught by U.S. Pat. No. 6,506,405,and U.S. Pat. No. 6,096,331 incorporated herein in their entirety.

Experiments on metabolic clearance rates done on cynomolgus monkeys(species Macaca fascicularis) have shown the safe use of large dosagesof elemental iron derived from iron dextran. (All experiments werepreformed in compliance with the Animal Welfare Act and Regulations.)Dosages of 400 mg and 500 mg of elemental iron, derived from irondextran, per kg of body weight were safely administered to thecynomolgus monkeys by intravenous infusion. The iron dextran showed aprotracted plasma residence time which functions as a decoy for thephagocytic system to redistribute the disclosed Composition to theplasma with few negative side effects. As shown in FIGS. 15A, B and C,the administered iron dextran remained in the monkey plasma for at least120 hours, at milligram levels. Single dosages of iron dextran were alsoseparately administered to monkeys, as shown in FIG. 16, with fewnegative side effects, i.e. abdominal swelling. The monkey model clearsthe iron dextran from the system much more very rapidly, as compared tohumans, because of a higher metabolic rate. Therefore, a longer plasmaresidence time is anticipated in humans, as has been shown in research,such as, for example, Henderson & Hillman, (1969).

The side effects of the Composition, with or without the addition of aniron dextran compound, are far fewer than the well-known side effects ofthe standardly administered chemotherapy, although the disclosedComposition can be used in conjunction with additional therapeuticagents. The disclosed Composition and iron dextran have breakdownbyproducts of copper and iron, which support the bioproduction of redblood cells, white blood cells and platelets. Because the Compositionsupports the hemopoietic system, its use limits or eliminates thewell-known devastating fatigue, risk of infection, and the adverseeffects of cytotoxic chemotherapy on the bone marrow (and other quicklygrowing cells) that are standardly caused by commonly used chemotherapyagents. In addition, the use of ancillary medications such as colonystimulating factors to accelerate bone marrow recovery anderythropoietin, a colony stimulating growth factor for red blood cellsfor the prevention of severe myelosuppression, and their severe sideeffects can be restricted. Since the need for the use of these drugs canbe restricted, the quality of life of the patient may be improved.

For diagnostic purposes, the Composition may be labeled with magnetictargeted carriers to allow imaging of the cancer cells and provideinformation to determine further medical treatments, including targetingtumors with external magnets. (Johnson, An Innovative Drug DeliveryTechnology, Magnetics Business & Technology Magazine, (2002)). A widevariety of other labels may be employed, such as radionuclides, fluors,enzymes, enzyme substrates, enzyme co-factors, enzyme inhibitors,ligands (particularly haptens), etc., and are well known to thoseskilled in the art.

Since the disclosed composition, iron dextran, and empty liposomes areall formed of biocompatible materials, all may be administered over anextended period of time as compared to other chemotherapeutic agents.The effective dose or effective amount can vary subject to theevaluation of the those of skill in the art in relation to theparticular type of cancer, the regimen of administration, the bodyweight of the subject, the aggressiveness of the cell growth and thedegree in which the subject has been negatively affected by priorchemotherapy. In general, a therapeutically effective amount is thatwhich decreases, or at minimum prevents further growth, of a primary ormetastatic tumor.

The disclosed Composition can be administered to a patient as apharmaceutical composition in combination with a pharmaceutical carrier.A pharmaceutical carrier can be any compatible, non-toxic substancesuitable for delivery of the Composition to the patient that ismedically acceptable. Sterile water, alcohol, fats, waxes, and inertsolids may be included in the carrier. Pharmaceutically acceptedadjuvants (buffering agents, dispersing agent) may also be incorporatedinto the pharmaceutical compound. In one embodiment, the Composition maybe combined with sterile water, or deinozed water and free dextran,dextran free of drug, to form a sterile colloidal suspension.

The disclosed Composition may be administered to a patient in a varietyof ways, such as oral, intravenous, subcutaneous, intraperitoneal,intrathecal, intramuscular, intracranial, inhalational, topical,transdermal, suppository (rectal), pessary (vaginal) or an implantablepolymer disclosed composition saturated depot or wafer, such as, forexample, a Giladel wafer®. Preferably, the pharmaceutical compound maybe administered parenterally, e.g., subcutaneously, intramuscularly orintravenously. Thus, the disclosed Composition may include a solutiondissolved in an acceptable carrier, preferably an aqueous carrier, forparenteral administration. A variety of aqueous carriers can be used,e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like.These solutions are sterile and generally free of particulate matter.These compounds may be sterilized by conventional, well-knownsterilization techniques. The Composition may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, and if necessaryfor sensitive patients, toxicity adjusting agents and the like, forexample sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate, etc. The concentration of the disclosedComposition in these formulations can vary widely, e.g., from less thanabout 0.1 mg to about 5 mg, ranging to as much as 10 mg or 15 mg or moreof the equivalent of elemental copper derived from the Composition perml of carrier. The preferred concentration of the disclosed Compositionis approximately 5 mg of the equivalent of elemental copper derived fromthe Composition per ml of carrier, and will be selected primarily basedon fluid volumes, viscosities, etc., in accordance with the particularmode of administration selected. The preferred pH range for use with thedisclosed Composition is between approximately 7 and approximately 8.5,and the more preferred pH range is between approximately 7.5 andapproximately 8.0.

Actual methods for preparing parenterally administerable compounds andadjustments necessary for administration to patients, typically mammals,will be known or apparent to those skilled in the art and are describedin more detail in, for example, Remington's Pharmaceutical Science: TheScience and Practice of Pharmacy, 20^(th) Ed., Lippincott, Williams &Wilkins; (2000), which is incorporated herein by reference.

It will be appreciated that the disclosed Composition addresses the verypressing problem of targeting cancer therapy for specificity, whilegreatly limiting or eliminating the horrendous side effects ofchemotherapy. Moreover, the disclosed Composition, especially when usedwith iron dextran, can overcome the difficulties of drug resistance. Thedisclosed composition may be employed with or without the iron dextranloading, to accomplish highly effective treatment against solid tumors,liquid tumors (blood), as well as metastatic cancers, while providing anagent that is cost effective because low dosages produce high activityand results. The disclosed Composition is designed to be administered byitself as an chemotherapeutic agent, with iron dextran, and/or inconjunction with conventional cancer therapies. Most importantly, theComposition's highly targeted and highly efficient cell kill rate cansave innumerable lives at a cost effective rate that can be madeavailable to any medical facility. For example, the disclosedComposition is very well suited to treat hepatocellular carcinoma, withor without iron loading. Hepatocellular carcinoma (“HCC”) is the mostcommon, primary cancer of the liver, and causes over 550,000 deathsannually, worldwide. Heretofore, no significantly effective treatmentsexisted for HCC. (Nakakura & Choti, Management of HepatocellularCarcinoma, Oncology, 14(7) (2000)). The disclosed Composition, however,may be introduced to the blood stream, and traffic through the hepaticartery to expose the normal hepatocytes and the cancerous hepatocytes tothe Composition. The hepatocytes breakdown the dextran to use or storeglucose as glycogen, and may also store copper and iron that is derivedfrom the Composition. Thus, the HCC cell is subject to the cytotoxicitycaused by the disclosed Composition. Any excess copper that is notstored, may be excreted through the biliary, and other bodily systems.Copper and iron from the hepatocytes are bound to the respective proteincarriers, which include transferrin and ceruloplamin to feed the cellsof the patient's body.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES Example 1

An in vitro human tumor screen was used to evaluate anti-proliferativeeffects of the disclosed Composition and the Composition in combinationwith the Base Compound of iron dextran. Human tumor cell linesrepresenting models of cancers with the greatest incidence, greatestincrease of incidence, the greatest mortality, or cancers that arehighly resistant to treatment were selected. The testing was conductedusing standard tissue culture techniques that are well known in the artand the ³H-thymidine assay for analysis.

Experimental Design.

This experiment was designed to evaluate the anti-proliferative andcytotoxic effects of the disclosed Composition alone, and in combinationwith Base Compound, and doxorubicin, also known by its trade nameAdriamycin, as a positive control which is a mainstay in the treatmentof many cancers used in combination with various chemotherapies (See,Chu and Devita, Cancer Chemotherapy Drug Manual 2003, Jones and BartlettPublishers, pg 138-139. (2003)) on the human tumor cell lines CAK-1renal, DLD-1 colon, LOX IMVI melanoma, MCF7 mammary, NCI-H23 lung,NCI-H460 lung, OVCAR-3 ovarian, PC-3 prostate, SNB-75 CNS, ZR-75-1mammary, and CEM-SS leukemic cells. See, FIG. 14. For all experiments,the cells were harvested, centrifuged to remove the media, and suspendedin fresh complete medium. Samples were taken to determine cell density.All cell counts were determined with a Coulter Model Z₁ cell counter(Beckman Coulter, Inc. Fullerton, Calif.) and viability was measuredwith propidium iodide staining followed by analysis on a Coulter EPICSXL flow cytometer (Beckman Coulter, Inc. Fullerton, Calif.). All celllines were each plated at 5×10³ cells per well in complete medium. Thefollowing day, the cells were dosed with 8 dilutions of the Compositionalone and the Composition in combination with the Base Compound of irondextran (60 μg/mL, which is the equivalent of elemental iron derivedfrom iron dextran). All iron dextran amounts are measured as theapproximate equivalent of elemental iron derived from the iron dextran.The Base Compound of iron dextran was also run alone as a control. Theplates were analyzed on Day 4 after the initiation of treatment.

The Composition was formed as follows: An inorganic copper salt, 4.854 gof copper nitrate (99.999%), was dissolved in 20 ml deionized water(Molecular Biology Reagent from Sigma-Aldrich), or distilled water couldalso be used. This solution was refluxed for approximately two hours.The copper salt solution was reacted with 2 g of oxidized dextran or 2 gof hydrogenated dextran at low temperature. (Clinical grade dextran,D4751 with an average molecular weight of 64,000-78,000, was purchasedfrom Sigma-Aldrich.) This solution was refluxed for 1 hour before adding0.2 ml of 0.5 M NaOH in the solution. After refluxing the solution foranother two hours, it was divided in half. Half of the solution wascombined with 2 g of oxidized dextran, and 40 ml of water were added,and followed by a two-hour refluxing step. The second half of thesolution was combined with hydrogenated dextran, 40 ml of water wereadded, and followed by a two-hour refluxing step. The solutions werethen each combined with 0.1 ml of 0.5 NaOH, and the reflux was continuedfor an additional two hours. The solutions were allowed to cool to roomtemperature. The resulting solution of a Cu(OH)₂-dextran nanoparticleswere precipitated in a controlled manner, wherein each Cu(OH)₂nanoparticle is covered by dextran molecules by adding 120 cc of 0.25 MNaOH to the final solutions. The water content of the solutions wasevaporated in a vacuum to increase the copper concentration in thesolutions. The precipitates with large particles were centrifuged toprepare the aqueous solutions of Cu(OH)₂-dextran nanoparticles. Thefinal copper concentration in the solutions was typically approximately5 mg/ml and the final pH ranges from approximately 7.5 to approximately8.5, and was assayed by atomic absorption spectrometry and/or inductivecoupled plasma spectrometry. The particle size of the Cu(OH)₂-dextrannanoparticles was determined by laser light scattering. The particlesize for oxidized dextran was in the range of approximately 150 nm toapproximately 200 nm and for hydrogenated dextran was in the range ofapproximately 20 nm to approximately 50 nm. After determining theparticle size, the solutions were tested for free copper ions using acopper electrode. The copper specific electrode was calibrated with fourknown copper concentrations solutions. These concentrations were asfollows: 0.1 moles/liter, 0.01 moles/liter, 0.001 moles/liter and 0.0002moles/liter (˜1 ppm). The millivolt readings of four standard Cu2+solutions were, respectively: Cu2+ Conc. mV 0.1 M 239 0.01M 206 0.001 M175 0.0002 M (1 ppm) 163The mV reading for these copper solutions was typically less than 130mV, which suggest that free Cu2+ concentration in solutions is less than1 ppm, and often lower than the level of detection. (As a point ofreference, the Environmental Protection Agency allows 1.3 ppm of copperin drinking water, see, for example, a website of the United StatesEnvironmental Protection Agency on safe water, and possible contaminantsof drinking water, including copper.) The colloidal suspensions of thedisclosed Composition in all samples had little free copper detected,typically approximately below the levels of detection of 1 ppm. Thecopper hydroxide solution prepared using oxidized dextran had a pH of8.5. The solution formed with hydrogenated dextran exhibited no freecopper ions, typically below the levels of detection of 1 ppm.

Preparation of Copper Hydroxide-Iron Hydroxide Nanoparticles(a) Preparation of Sample 1

A copper salt, 2.428 g, of Cu nitrate (99.999% pure, Alfa Aesar, catalog#10699) was combined with 0.2 g of FeCl₃, 6H₂O (purity 97-102%, AlfaAesar, Catalog #12497), and 4.0 g of hydrogenated dextran. Thesecomponents were dissolved in 70 ml of deionized water (Molecular BiologyReagent from Sigma-Aldrich). This solution was then refluxed forapproximately 3 hrs. The solution was allowed to cool before adding 92.8cc of 0.25M NaOH (Fisher ACS, catalog #S318-3) into the solution. Thefinal pH of the solution was 8.5. After 6 days, pH decreased to 6.85,and 1.7 cc of 0.25M NaOH solution was added to adjust the pH to 8.5.Analysis of the copper and iron concentration in solution was done byatomic absorption spectrometry (“M”) and/inductive coupled plasmaspectrometry (“ICP”). The solution was syringe filtered, and the darkgreen solution was stored in sterile vials. Iron oxyhydroxide may alsobe employed as a substitute for iron hydroxide in this or any sample.

(b) Preparation of Sample 2

The copper salt, 2.428 g, of Cu nitrate (99.999% pure, Alfa Aesar,catalog #10699) was combined with 0.4 g of FeCl₃, 6H₂O (purity 97-102%,Alfa Aesar, Catalog #12497), and 4.2 g of hydrogenated dextran. Thesecomponents were dissolved in 75 ml of deionized water (Molecular BiologyReagent from Sigma-Aldrich). This solution was refluxed forapproximately 3 hrs. The solution was allowed to cool before adding102.2 cc of 0.25M NaOH (Fisher ACS, catalog #S318-3) in the solution.The final pH of the solution was 8.5. After 6 days, pH decreased to 7.4,and 1.6 cc of 0.25M NaOH solution was added to adjust the pH to 8.5.Analysis of the copper and iron concentration in solution was done by AAand/ICP. The solution was centrifuged, and the dark green solution withslight haze was stored in sterile vials.

(c) Preparation of Sample 3

The copper salt, 2.428 g, of Cu nitrate (99.999% pure, Alfa Aesar,catalog #10699) was combined with 0.2 g of FeCl₃, 6H₂O (purity 97-102%,Alfa Aesar, Catalog #12497), 1.2 g of hydrogenated dextran, and 2.8 gdextran (MW=15,000). These components were dissolved in 70 ml ofdeionized water (Molecular Biology Reagent from Sigma-Aldrich). Thissolution was refluxed for approximately 3 hrs. The solution was allowedto cool before adding 83.2 cc of 0.25M NaOH (Fisher ACS, catalog#S318-3) into the solution. The final pH of the solution was 8.5. After6 days, pH decreased to 7.64, and 0.6 cc of 0.25M NaOH solution wasadded to adjust the pH to 8.5. Analysis of the copper and ironconcentration in solution was done by AA and/ICP. The solution wascentrifuged, and the dark green solution was stored in sterile vials.

Cell Lines and Standard Agents

The cell lines were propagated using standard tissue culture proceduresand seeded in microtiter plates prior to dosing. The control groupsincluded a Base Compound (60 μg/mL) only treatment, complete mediumcontrol, and positive control (doxorubicin, 1 μM). For eachconcentration level of the Composition, eight replicates of each cellline were treated.

Cell Culture

The cell lines used in the following Examples are listed below inChart 1. The Composition was tested on the listed solid tumors, andliquid tumors, but may be effectively used for any type of cancers. Thecell lines were propagated under sterile conditions and incubated at 37°C. in HEPA-filtered CO₂ tissue culture incubators with 5% CO₂ and 95%humidity. Each cell line was sub-cultured weekly to bi-weekly or morefrequently for use in experiments.

³H (Tritiated)-Thymidine Assay

Anticellular effects of the compounds on the tumor lines were assessedwith the ³H-thymidine DNA incorporation assay. Tritiated-thymidine waspurchased as a 1 mCi stock and diluted 1:25 in media. One day prior toharvest, 25 μL (1 μCi) of the diluted ³H-thymidine was added to eachwell, and the plates were incubated overnight. The following morning thecells were harvested onto glass fiber filters using a Skatron cellharvester (Molecular Devices Corporation, Sunnyvale Calif.). The filterswere then placed in scintillation vials and scintillation cocktail wasadded (Beckman Coulter, Inc. Fullerton, Calif.). The vials were thenread on a Beckman LS6000IC liquid scintillation counter (BeckmanCoulter, Inc. Fullerton, Calif.) and the data were reported as countsper minute (CPM). The data were transferred into Lotus 123 forprocessing.

For all cell lines, the cells were harvested, centrifuged to remove themedia, and suspended in fresh complete medium. Samples were taken todetermine cell density. The cell count was determined with a CoulterModel Z₁ cell counter (Beckman Coulter, Inc. Fullerton, Calif.) and cellviability was measured with propidium iodide staining. Analysis was thenconducted on a Coulter EPICS XL flow cytometer (Beckman Coulter, Inc.Fullerton, Calif.). The cell lines were each plated at 5×10³ cells perwell in complete medium. On the second day, the cells were washed with 8dilutions of the disclosed Composition alone, or in combination with theBase Compound at the concentration of 60 μg/mL. A control was run bywashing cells with only the Base Compound. On day 4 after the initialtreatment, the plates were analyzed. The results were summarized below:TABLE 1 IC₅₀ (μg/mL) IC₅₀ (μg/mL) Composition and Cell Line CompositionBase Compound (60 μg/mL) CAKI-1 renal 1.440 1.138 DLD-1 colon 1.4300.196 NCI-H23 lung >10 1.718 NCI-H 460 lung 1.183 0.131 LOX IMVImelanoma 6.718 0.513 MCF7 mammary 2.213 0.972 OVCAR-3 ovarian 3.6620.299 PC-3 prostate >10 1.869 SNB-75 CNS 0.895 0.095 ZR-75-1 mammary >102.031 CEM-SS Leukemic 1 5.87 CEM-SS Leukemic 2 4.975

The experiments, described below, performed on tumor cells lines arepresented with results in Table 1, with the exception of the HT29humancolon adenocarcinoma cells. The Composition plus the Base Compound at 60μg/ml resulted in 100% cell kill, with the exception of the CAKI-1 renalline, which resulted in 99% cell kill. Moreover, the further addition ofincreased base compound to composition increases the cytotoxicity, ifnecessary. In three cell lines that were resistant to Composition alone,up to 10 μg/ml, namely NCI-H23 lung, ZR-75-1 mammary and PC-3 prostate,resistance was completely overcome with the addition of Base Compound tothe Composition, at 60 μg/ml, resulting in 100% cell kill. For all celllines that were exposed to the Base Compound, the IC₅₀ was loweredsignificantly by the synergistic, ctyotoxic effects of the Base Compoundin combination with the disclosed Composition, demonstrating enhancedcell kill with the addition of Base Compound. For all the cell linesthat were exposed to the Base Compound, Composition with the BaseCompound equaled or exceeded the cell kill of doxorubicin, a mainstaychemotherapeutic drug in the treatment of breast cancer and othercancers, which is well known to have many severe side effects.

Example 2

FIG. 1 The Release of ROS (reactive oxygen species) by HT29 Human ColonAdenocarcinoma Cell Line After 24-Hr Incubation.

The data were obtained after a 24 hour incubation of HT29 cells with 10μg/mL of the disclosed Composition, 60 μg/mL of the Composition plusBase Compound, and 60 μg/mL of the iron dextran Base Compound alone. Theassay depends on a non-fluorescent substrate added to wells in whichcells are growing. Where ROS are present, the substrate is broken downto form a fluorescent product. The data in FIG. 1 demonstrates that theComposition produces ROS above the level of the control of fresh mediumand the Base Compound. The data further demonstrates an increasedproduction of ROS with the disclosed Composition in combination with theBase Compound, above that of the disclosed Composition or the BaseCompound alone.

The combination of the disclosed Composition and the Base Compoundgenerates a significant amount of ROS, as do radiation treatments forcancer patients, which is generally believed to exert its cytotoxiceffect by the generation of DNA damaging free radicals. The combinationof the disclosed Composition and the Base Compound can be used inconjunction with radiation treatment can increase the amount of cancerkilling free radicals generated by radiation and exert increasedcell-kill over radiation alone. This is known in the art as a radiosensitizer, compounds which amplify and potentiate the cytotoxic effectof radiation.

Example 3

FIG. 2A discloses a graph of the mean inhibitory concentration of thedisclosed Composition against the NCI-H23 lung cells. The inhibitoryconcentration 50 (“IC₅₀”) is defined as the concentration of theemployed composition or compound that is inhibitory or effective on 50%,or more, of the cells used in an experimental procedure. The disclosedComposition has a highly effective IC₅₀ level of approximately 10 μg/mlwhen applied to NCI-H23 lung cells. FIG. 2B provides the absorbancevalues of the disclosed Composition, the Base Compound, doxorubicin, anda control for the NCI-H23 Lung cells in both media and MTS reagent(Promega, Madison Wis., U.S.). The MTS reagent is a tetrazolium saltthat it is converted to a colored compound of formazan when applied tolive cells, with the emission of light at approximately 490 nm. Thedisclosed Composition inhibited forty percent of the cultured NCI-H23Lung cells at a dosage 10 μg/mL. Although doxorubicin exhibited a highinhibitory effect, it is also known to have many detrimental sideeffects when used in vivo, which the disclosed Composition will notcause. The absorbance value units are also given and some backgroundabsorbance was assumed to have occurred, and typically ranges between0.2-0.4 units after 4 hours of incubation. FIG. 2C discloses theexpected theoretical absorbance levels of the disclosed Composition forvarying IC levels.

As shown in FIG. 2D, the NCI-H23 lung cells showed little or noresistance to both the 3 μg/mL and 10 μg/mL dosages of the Compositionwith the addition of the Base Compound. This combination of theComposition with the addition of the Base Compound resulted in over a99-100% inhibition of the cells in vitro, which equals that ofdoxirubicin. The concentration of the Composition together with the BaseCompound was 60 μg/mL. FIG. 2E shows the absorbance values andinhibition percentages of the Composition plus Base Compoundcombination, which demonstrated 100% inhibition of the NCI-H23 lungcells at the low dosage of 10 μg/mL. FIG. 2F show the statisticalresults of the regression output for the experiments.

Example 4

FIG. 3A shows over 90% inhibition of NCI-H460 lung cells with the highactivity and cytotoxicity of the disclosed Composition at a 10 μg/mLconcentration. The disclosed Composition was also highly effective at a3 μg/mL concentration with a 90% inhibition rate and nearly 50%inhibition of the cells at only a 1 μg/mL concentration. The disclosedComposition also exhibited significant inhibition percentages at verylow dosages. FIG. 3B provides the absorbance value units from thevarying dosages, as shown, as well as the inhibition percentages for thedifferent dosages, which were very high. FIG. 3C discloses the IC₅₀ at alow dosage of 1.183 μg/mL of the Composition, and the statisticalanalysis of the regression output.

This example examines the effect of toxicity of the Composition plus theBase Compound against NCI-H460 lung cells. The results of these testsare shown in FIGS. 3D, 3E and 3F. FIG. 3D shows an enhanced cell kill ofthe NCI-H460 lung cells where the Base Compound is added to thedisclosed Composition, as compared to the results of the Compositionitself. As shown in FIG. 3A, 10 μg/ml of the Composition were appliedfor a resulting 100% cell kill. Where the Base Compound was added to theComposition, 1 μg/ml of Composition plus Base Compound resulted in a100% cell kill, as shown in FIG. 3D. The concentration of Compositionplus Base Compound was a very efficient 0.131 μg/ml resulting in an IC₅₀inhibition, and by contrast, the concentration of the Composition alonewas 1.183 μg/ml to resulting in an IC₅₀ inhibition of the experimentalcells. FIG. 3E discloses the absorbance value units from the varyingdosages, as shown, as well as the inhibition percentages for thedifferent dosages, which were very high. The combination of theComposition with the Base Compound was shown to be highly effective inits toxic activity against NCI-H460 Lung cells.

Example 5

This example examines the effect of toxicity of the Composition aloneagainst MCF7 mammary cells. FIG. 4A shows the very high activity of thedisclosed Composition against MCF7 mammary cells. The Compositionexhibited over 90% inhibition of the cells at 10 μg/mL, and over 60%inhibition at 3 μg/mL. FIG. 4B provides the absorbance values fordisclosed Composition, plus the media and MTS. FIG. 4C provides thecalculated IC₅₀ of 2.213 μg/mL, and the regression output for 3.000 and1.000 concentrations.

FIGS. 4D, 4E and 4F examine the effect of toxicity of the Composition incombination with the Base Compound against MCF7 mammary cells. Thesetests show an enhanced cell kill with the addition of the Base Compoundto this cell line, as compared to the disclosed Composition only, asshown in FIGS. 4A, 4B, and 4C. FIG. 4A shows that 10 μg/ml were requiredfor 90% cell kill. When tested in combination with the Base Compound,only 3 μg/ml of the Composition is required for 100% of cell kill, whichlowered the IC₅₀ to 0.972 μg/ml for the same cell line.

Example 6

FIG. 5A graphs the effect of toxicity of the disclosed Compositionagainst ZR-75-1 mammary cells. These tests showed an approximately 35%inhibition at 10 μg/mL of the ZR-75-1 mammary cells. This cell lineshowed resistance to the Composition at concentrations up toapproximately 10 μg/ml. The absorbance values and inhibition percentagesare shown in FIGS. 5B and 5C.

FIG. 5D discloses the very high activity of the combination of thedisclosed Composition and the Base Compound against the ZR-75-1 mammarycells. The IC₅₀ of this combination was found to be a surprisingconcentration and calculated to approximately 2.031 μg/mL. Theresistance of ZR-75-1 mammary cells was essentially eliminated with theaddition of the Base Compound to the Composition. The 10 μg/ml of theComposition plus the Base Compound resulted in an approximately 100%cell kill for this cell line, a very effective therapeutic with few sideeffects or negative aspects. FIG. 5E provides the absorbance values andinhibition percentages of this experiment with significant inhibition at3 μg/ml and 10 μg/ml dosages. FIG. 5F discloses a calculated IC₅₀ rateof a low concentration of approximately 2.031 μg/ml, and the regressionoutput for the experiment.

Example 7

FIG. 6A shows the results of toxicity tests of the Composition on PC-3prostate cells. The PC-3 prostate cells exhibited resistance to theComposition up to concentrations of approximately 10 μg/mL, with somecellular inhibition at 0.01 μg/mL. The dosage of 10 μg/mL resulted in a17% inhibition of the prostate cells. FIGS. 6B and 6C provide theabsorbance values and statistical results of the experiment ofComposition on prostate cells.

FIG. 6D shows the effects of toxicity of the Composition plus the BaseCompound against PC-3 prostate cells. The resistance of PC-3 prostatecells is essentially eliminated with the addition of Base Compound. Theaddition of the Base Compound shows an enhanced cell kill in these teststo this cell line, as compared to the Composition alone in FIG. 6A. Aconcentration of 10 μg/ml of Composition in combination with the BaseCompound resulted in a 100% of cell kill, with an IC₅₀ that wasextremely low at a concentration of 1.869 μg/ml. Concentrations as lowas 3 μg/ml resulted in approximately 90% inhibition of the cell line.FIGS. 6E and 6F provide the absorbance value and statistical results ofthis experiment.

The cause of the aberrant experimental results found in both FIGS. 6Aand 6D at the 0.01 μg/ml concentration level was not determined.

Example 8

FIG. 7A shows the high toxicity effect of the Composition on DLD-1 coloncells. The Composition displayed significant cell kill rates at allconcentrations, including at very low concentrations. The resultinginhibition percentages, as shown in FIG. 7B, were very high with a 95%inhibition of the DLD-1 colon cells with 10 μg/mL of the Composition.FIG. 7C provides the statistical analysis of the experimental results.

FIG. 7D provides the results of toxicity experiments with theComposition in combination with the Base Compound on DLD-1 colon cells.These tests showed an enhanced cell kill with the addition of BaseCompound as compared to the Composition alone. As shown in FIGS. 7D and7E, an exceedingly low concentration of 3 μg/ml of Composition plus BaseCompound was required for 100% of cell kill, as compared to a 95% cellkill by 10 μg/ml of the Composition alone, shown in FIGS. 7A and 7B. TheIC₅₀ was lowered with the addition of Base Compound for the same cellline to 0.196 μg/ml from an IC₅₀ of 1.430 μg/ml for the Compositionalone.

Example 9

FIG. 8A discloses the highly toxic effect of the Composition againstOVCAR-3 ovarian cells with over 90% inhibition rate at very lowconcentrations of 1 μg/mL, 3 μg/mL and 10 μg/mL. The absorbance valuesand statistical results of these experiments are given in FIGS. 8B and8C.

The toxicity effects of the Composition in combination with the BaseCompound on OVCAR-3 ovarian cells are shown in FIG. 8D. These testsshowed an enhanced cell kill with the addition of the Base Compound ascompared to Composition alone. The combination of the Composition withthe Base Compound resulted in a 100% cell kill at the concentration of 3μg/ml, whereas the application of the Composition alone required 10μg/ml for a resulting 95% cell kill. The IC₅₀ for the combination of theComposition and the Base Compound was lowered to the very lowconcentration of 0.299 μg/mL.

Example 10

The toxicity effects of the Composition on CAKI-1 renal cells are shownin FIG. 9A. The Composition showed very high activity against this cellline, even at low dosages. The inhibition percentages showed significantactivity of the Composition at concentrations as low as 0.01 μg/mL for20.3% inhibition, and 83.6% inhibition of the cell line at theconcentration of 10 μg/ml. See, FIGS. 9B and 9C.

The combination of the Composition plus the Base Compound showed veryhigh activity against CAKI-1 renal cells, as shown in FIG. 9D. Thesetests show an enhanced cell kill with the addition of Base Compound ascompared to the use of the Composition alone as shown in FIG. 9A. Aconcentration of 10 μg/ml of the Composition resulted in a 99% cellkill. The IC₅₀ was lowered with the addition of Base Compound to 1.138μg/mL for this cell line in contrast to the IC₅₀ of Composition alone,which was 1.44 μg/mL. In the experiments on the CAKI-1 renal cells, boththe Composition and the Composition plus the Base Compound demonstratedvery significant activity with low IC₅₀ rates.

Example 11

FIG. 10A shows the toxic effect the Composition against LOX IMVImelanoma cells. The experiment showed high activity of the Compositionand resulted in an approximately 82% inhibition of the cell line at aconcentration of 10 μg/mL. FIG. 10B shows the absorbance rates and theinhibition percentages of the experiments with some inhibition at 3μg/mL. FIG. 10C provides the statistical analysis of the results,including a calculated IC₅₀ of 6.718 μg/mL.

FIG. 10D shows the high activity of the Composition plus the BaseCompound on LOX IMVI melanoma cells. The Composition in combination withthe Base Compound had highly toxic effects on this cell line, includingat very low dosages. These tests show an enhanced cell kill with theaddition of Base Compound to this cell line as compared to the use ofComposition alone, as shown in FIG. 10A. A 3 μg/ml concentration of theComposition resulted in 100% cell kill, whereas 10 μg/ml were requiredfor 82% cell kill with the Composition alone, as shown as FIG. 10A. TheIC₅₀ of Composition alone was 6.718 μg/mL, the IC₅₀ was lowered with theaddition of the Base Compound for the same cell line to 0.513 μg/mL.

Example 12

The toxicity of the Composition was tested against SBN-75 CNS cells. Theresults are shown in FIG. 11A, and show very high activity of theComposition. A concentration of 10 μg/mL resulted in a 100% inhibitionof the SBN-75 CNS cells, and a concentration of only 3 μg/mL resulted inan approximately 85% inhibition of this cell line. FIGS. 11B and 11Cprovide the absorbance values and the statistical analysis of theresults.

FIG. 11D discloses the high toxicity effects of the Composition plus theBase Compound against SBN-75 CNS cells. The combination of theComposition and the Base Compound resulted in a very successful 100%inhibition rate at dosages of 1 μg/mL, 3 μg/mL, and 10 μg/mL. Thesetests show an enhanced cell kill with the addition of the Base Compoundto this cell line as compared to the use of the Composition alone. Aconcentration of 1 μg/ml of Composition plus Base Compound resulted in100% of cell kill, as compared to a concentration of 10 μg/ml of theComposition alone for 100% cell kill. The IC₅₀ was lowered with theaddition of Base Compound for the same cell line to 0.095 μg/ml.

Example 13

The CEM-SS cells were obtained from the AIDS Research and ReferencesReagent Repository (Bethesda, Md.). These cells were passaged in T-75flasks in tissue culture media, which included RPMI 1640 medium (nophenol red), with 10% fetal bovine serum (heat inactivated), 2 mML-glutamine, 100 U/mL penicillin, 100 μg/ml streptomycin, and 10 μg/mlgentamycin. One day preceding the tritated thymidine assay, the cellswere split 1:2 to assure that they were in an exponential growth phaseat the time of the cytotoxicty tests. On the day of the assay, the cellswere collected by centrifugation, washed twice with tissue culturemedium, above, and resuspended at 5×10⁴ cells per mL, and resuspended infresh tissue culture medium. The total cell and viability counting wasperformed with a hemacytometer. Cell viability prior to the assay wasdetermined by trypan blue dye exclusion and exceeded, as it must 95%.Cultures were incubated for 6 days at 37° C., 5% CO₂.

The highly toxic effects of the Composition alone against CEM-SSleukemic cells are shown in FIGS. 12A, 12 B, 12C. These Figures show anIC₅₀ of 5.87 μg/mL and a highly efficient cell kill rate ofapproximately 98% at a dosage of 10 μg/mL.

Example 14

The high activity of the Composition alone against CEM-SS leukemic cellsare shown in FIGS. 13A, 13 B, 13C. These Figures show IC₅₀ of 4.975μg/mL and a highly efficient cell kill rate of approximately 100% at adosage of 10 μg/mL.

In the foregoing description, certain terms are used to illustrate thepreferred embodiments. However, no unnecessary limitations are to beconstrued by the terms used, since the terms are exemplary only, and arenot meant to limit the scope of the present invention.

It is further known that other modifications may be made to the presentinvention, without departing from the scope of the invention, as notedin the appended Claims.

1-107. (canceled)
 108. A composition for use as a pharmaceutical fortargeting at least one pre-cancerous affecting virus, for prevention ofcancer comprising: a core essentially of biologically acceptable fixedcopper compound, and a sheath that encapsulates the biologicallyacceptable fixed copper compound.
 109. The composition as in claim 108where said virus is a hepatocellular affecting virus and said cancer isliver cancer.
 110. The composition of claim 108, wherein the compositionis used for therapeutic treatment of hepatitis B virus.
 111. Thecomposition of claim 108, wherein the composition is used for preventivepre-acute stage treatment of hepatitis B viral infection.
 112. Thecomposition of claim 108, wherein the composition is used fortherapeutic treatment of hepatitis C virus.
 113. The composition ofclaim 108, wherein the composition is used for preventive pre-acutestage treatment of hepatitis C viral infection.
 114. The composition asin claim 108 wherein said virus is human papilloma virus (HPV) and saidcancer is cervical cancer.
 115. The composition as in claim 108 whereinsaid virus is Epstein-Barr Virus (EBV) and said cancer is Burkitt'slymphoma.
 116. The composition as in claim 108 wherein said virus isHuman T-cell Lymphotropic Virus (HTLV-1) and said cancer is Adult T-cellLeukemia/Lymphoma.
 117. The composition of claim 108, wherein the sheathis formed of a material selected from the group consisting essentiallyof lipids, polypeptides, oligopeptides, polynucleotides, proteins,liposomes and combinations thereof.
 118. The composition of claim 108,wherein the sheath is formed of a material selected from the groupconsisting essentially of a glucose, a saccharide, a polysaccharide, adextran, liposomes, derivatives and combinations thereof.
 119. Thecomposition of claims 108, wherein a liposome coat encapsulates thesheath.
 120. The composition of claim 108, wherein a polysaccharidesheath encapsulates the composition.
 121. The composition of claim 108,wherein the biologically acceptable fixed copper compound is selectedfrom the group consisting of copper hydroxide, copper oxide, copperoxychloride, copper carbonate basic, copper sulfate, copper-ironhydroxide, copper-iron oxide, copper-iron oxyhydroxide, copper sulfatebasic, cuprous oxide, cupric hydroxide-iron hydroxide, cupric citrate,cupric glycinate, cupric gluconate, cupric phosphate, cuprobam, cupricsalicylite, indigo copper, cupro-cupric sulfate, cuprous sulfate,cuprous sulfate hemihydrite, brochantite, langite, malachite, azurite,cheesylite, cornetite, dihydyrite, libethenite, phosphorochalcite,pseudolibethenite, pseudo-malachite, tagilite, antlerite, covellite,marshite, cuprite, chalcocite, Rogojski's salt, brochantite,hydrocyanite, chalcanthtite, nantokite and dolerophane.
 122. A chemicalcomposition for use in targeting at least one pre-cancerous affectingvirus, for prevention of cancer comprising: a core essentially of afixed copper-iron compound, and a sheath that encapsulates the core ofthe copper-iron compound.
 123. The composition as in claim 122 wheresaid virus is a hepatocellular affecting virus and said cancer is livercancer.
 124. The composition of claim 122, wherein the composition isused for therapeutic treatment of hepatitis B virus.
 125. Thecomposition of claim 122, wherein the composition is used for preventivepre-acute stage treatment of hepatitis B viral infection.
 126. Thecomposition of claim 122, wherein the composition is used fortherapeutic treatment of hepatitis C virus.
 127. The composition ofclaim 122, wherein the composition is used for preventive pre-acutestage treatment of hepatitis C viral infection.
 128. The composition asin claim 122, wherein said virus is human papilloma virus (HPV) and saidcancer is cervical cancer.
 129. The composition as in claim 122, whereinsaid virus is Epstein-Barr Virus (EBV) and said cancer is Burkitt'slymphoma.
 130. The composition as in claim 122, wherein said virus isHuman T-cell Lymphotropic Virus (HTLV-1) and said cancer is Adult T-cellLeukemia/Lymphoma.
 131. The composition of claim 122, wherein the sheathis formed of a material selected from the group consisting essentiallyof a glucose, a saccharide, a polysaccharide, a dextran, liposomes,derivatives and combinations thereof.
 132. The composition of claim 122,wherein the sheath is formed of a material selected from the groupconsisting essentially of lipids, polypeptides, oligopeptides,polynucleotides, proteins, and combinations thereof.
 133. Thecomposition of claims 122, wherein a liposome coat encapsulates thesheath.
 134. The composition of claim 122, wherein the fixed copper-ironcompound core is copper hydroxide-iron hydroxide.
 135. The compositionof claim 122, wherein said fixed copper-iron compound core is copperhydroxide-iron oxide.
 136. The composition of claim 122, wherein saidfixed copper-iron compound core is copper-iron oxyhydroxide.
 137. Thecomposition of claim 122, wherein the core particle is targeted with atargeting agent.
 138. The composition of claim 122, wherein the coreparticle is targeted with a marker.
 139. The composition of claim 122,wherein the core particle is targeted with magnetic particles.
 140. Achemical composition for targeting at least one pre-cancerous affectingvirus, for prevention of cancer comprising: a compound of synergisticcomponents of a fixed copper and iron to form a core particle.
 141. Thecomposition as in claim 140 where said virus is a hepatocellularaffecting virus and said cancer is liver cancer.
 142. The composition ofclaim 140, wherein the composition is used for therapeutic treatment ofhepatitis B virus.
 143. The composition of claim 140, wherein thecomposition is used for preventive pre-acute stage treatment ofhepatitis B viral infection.
 144. The composition of claim 140, whereinthe composition is used for therapeutic treatment of hepatitis C virus.145. The composition of claim 140, wherein the composition is used forpreventive pre-acute stage treatment of hepatitis C viral infection.146. The composition as in claim 140 wherein said virus is humanpapilloma virus (HPV) and said cancer is cervical cancer.
 147. Thecomposition as in claim 140 wherein said virus is Epstein-Barr Virus(EBV) and said cancer is Burkitt's lymphoma.
 148. The composition as inclaim 140 wherein said virus is Human T-cell Lymphotropic Virus (HTLV-1)and said cancer is Adult T-cell Leukemia/Lymphoma.
 149. The compositionof claim 140, wherein the copper of the core particle is selected fromthe group consisting of copper hydroxide, copper oxide, copperoxychloride, copper carbonate basic, copper sulfate, copper-ironhydroxide, copper-iron oxide, copper-iron oxyhydroxide, copper sulfatebasic, cuprous oxide, cupric hydroxide-iron hydroxide, cupric citrate,cupric glycinate, cupric gluconate, cupric phosphate, cuprobam, cupricsalicylite, indigo copper, cupro-cupric sulfate, cuprous sulfate,cuprous sulfate hemihydrite, brochantite, langite, malachite, azurite,cheesylite, cornetite, dihydyrite, libethenite, phosphorochalcite,pseudolibethenite, pseudo-malachite, tagilite, antlerite, covellite,marshite, cuprite, chalcocite, Rogojski's salt, brochantite,hydrocyanite, chalcanthtite, nantokite and dolerophane.
 150. Thecomposition of claim 140, wherein the iron of the core particle is ironhydroxide.
 151. The composition of claim 140, wherein the iron of thecore particle is iron oxide.
 152. The composition of claim 140, whereinthe iron of the core particle is an iron compound.
 153. The compositionof claim 140, wherein the core particle is encapsulated with a sheath.154. The composition of claim 140, wherein the core particle is coatedwith liposomes.
 155. The composition of claim 140, wherein the coreparticle is targeted with a targeting agent.
 156. The composition ofclaim 140, wherein the core particle is targeted with a marker.
 157. Thecomposition of claim 140, wherein the core particle is targeted withmagnetic particles.
 158. A composition for medicinal use in mammalscomprising: a biologically acceptable, fixed copper compound core, andsaid fixed copper compound core being encapsulated, encoated, adsorbed,complexed or bound in at least one of a sheath, a shell, a polymericshell, a cover, a casing, an encoating, a jacket or combination thereof,and a pharmaceutically acceptable carrier, said sheath, shell, polymericshell, cover, casing, encoating, jacket or combination thereof beingmade of a material targeting at least one pre-cancerous affecting virus,and preventing immediate chemical interaction of the core with thesurrounding cellular environment; said fixed copper compound corefurther comprising a material that remains in circulation of themammals, wherein said targeting of said virus is used for prevention ofcancer.
 159. The composition as in claim 158 where said virus is ahepatocellular affecting virus and said cancer is liver cancer.
 160. Thecomposition of claim 158, wherein the composition is used fortherapeutic treatment of hepatitis B virus.
 161. The composition ofclaim 158, wherein the composition is used for preventive pre-acutestage treatment of hepatitis B viral infection.
 162. The composition ofclaim 158, wherein the composition is used for therapeutic treatment ofhepatitis C virus.
 163. The composition of claim 158, wherein thecomposition is used for preventive pre-acute stage treatment ofhepatitis C viral infection.
 164. The composition of claim 158, whereinthe composition is used for therapeutic treatment of pre-cancerous viralinfection.
 165. The composition of claim 158, wherein the composition isused for destroying Hepatitis B virus particles in a mammal infectedwith Hepatitis B prior to the onset of liver cancer in said infectedmammal.
 166. The composition of claim 158, wherein the composition isused for destroying Hepatitis C virus particles in a mammal infectedwith Hepatitis C prior to the onset of liver cancer in said infectedmammal.
 167. The composition as in claim 158 wherein said virus is humanpapilloma virus (HPV) and said cancer is cervical cancer.
 168. Thecomposition as in claim 158 wherein said virus is Epstein-Barr Virus andsaid cancer is Burkitt's lymphoma.
 169. The composition as in claim 158wherein said virus is Human T-cell Lymphotropic Virus (HTLV-1) and saidcancer is Adult T-cell Leukemia/Lymphoma.
 170. A chemical compositionfor treating at least one pre-cancerous affecting virus causing cancerin mammals comprising: a compound of synergistic components of a fixedcompound of copper and iron to form a core particle, said core particletargeting said virus without affecting the surrounding environment. 171.The composition as in claim 170 where said virus is a hepatocellularaffecting virus and said cancer is liver cancer.
 172. The composition asin claim 170 wherein said affecting virus is selected from the groupconsisting of Hepatitis B virus and Hepatitis C virus.
 173. Thecomposition as in claim 170 wherein said virus is human papilloma virus(HPV) and said cancer is cervical cancer.
 174. The composition as inclaim 170 wherein said virus is Epstein-Barr Virus (EBV) and said canceris Burkitt's lymphoma.
 175. The composition as in claim 170 wherein saidvirus is Human T-cell Lymphotropic Virus (HTLV-1) and said cancer isAdult T-cell Leukemia/Lymphoma.
 176. A method for treating precancerousVirus infection and prevention of cancer in a patient comprising:forming a colloidal solution composition of at least a fixed coppercompound core, and said fixed copper compound core being encapsulated,encoated, adsorbed, complexed or bound in at least one of a sheath, ashell, a polymeric shell, a cover, a casing, an encoating, a jacket orcombination thereof, and a pharmaceutically acceptable carrier, saidsheath, shell, polymeric shell, cover, casing, encoating, jacket orcombination thereof being made of a material targeting at least oneprecancerous virus, and preventing immediate chemical interaction of thecore with the surrounding cellular environment; said fixed coppercompound core further comprising a material that remains in circulationof the mammals, wherein said targeting of said virus is used forprevention of cancer.
 177. The composition as in claim 176 where saidvirus is a hepatocellular affecting virus and said cancer is livercancer.
 178. The method for treating precancerous hepatocellular Virusinfection and prevention of liver cancer of claim 176, wherein thecomposition is used for therapeutic treatment of hepatitis B virus. 179.The method for treating precancerous hepatocellular Virus infection andprevention of liver cancer of claim 176, wherein the composition is usedfor preventive pre-acute stage treatment of hepatitis B viral infection.180. The method for treating precancerous hepatocellular Virus infectionand prevention of liver cancer of claim 176, wherein the composition isused for therapeutic treatment of hepatitis C virus.
 181. The method fortreating precancerous hepatocellular Virus infection and prevention ofliver cancer of claim 176, wherein the composition is used forpreventive pre-acute stage treatment of hepatitis C viral infection.182. The method for treating precancerous hepatocellular Virus infectionand prevention of liver cancer of claim 176, wherein the composition isused for therapeutic treatment of pre-cancerous viral infection. 183.The method for treating precancerous hepatocellular Virus infection andprevention of liver cancer of claim 176, wherein the composition is usedfor destroying Hepatitis B virus particles in a mammal infected withHepatitis B prior to the onset of liver cancer in said infected mammal.184. The method for treating precancerous hepatocellular Virus infectionand prevention of liver cancer of claim 176, wherein the composition isused for destroying Hepatitis C virus particles in a mammal infectedwith Hepatitis C prior to the onset of liver cancer in said infectedmammal.
 185. The composition as in claim 176 wherein said virus is humanpapilloma virus (HPV) and said cancer is cervical cancer.
 186. Thecomposition as in claim 176 wherein said virus is Epstein-Barr Virus(EBV) and said cancer is Burkitt's lymphoma.
 187. The composition as inclaim 176 wherein said virus is Human T-cell Lymphotropic Virus (HTLV-1)and said cancer is Adult T-cell Leukemia/Lymphoma.
 188. The method ofclaim 176 further comprising the steps of administering the colloidalsolution composition to a patient; monitoring the presence of saidhepatocellular virus in the patient; and re-administering thecomposition at intervals based on results of the monitoring.
 189. Amethod for treating an affecting virus and preventing cancer comprisingthe steps of administering a compound of synergistic components of fixedcopper and fixed iron to a patient infecting with at least onepre-cancerous virus.
 190. The composition as in claim 189 where saidvirus is a hepatocellular affecting virus and said cancer is livercancer.
 191. The method as in claim 189 wherein said hepatocellularaffecting virus is selected from the group consisting of Hepatitis Bvirus and Hepatitis C virus.
 192. The composition as in claim 189wherein said virus is human papilloma virus (HPV) and said cancer iscervical cancer.
 193. The composition as in claim 189 wherein said virusis Epstein-Barr Virus (EBV) and said cancer is Burkitt's lymphoma. 194.The composition as in claim 189 wherein said virus is Human T-cellLymphotropic Virus (HTLV-1) and said cancer is Adult T-cellLeukemia/Lymphoma.